Modulation of Receptor Number in Cultured Cells

培养细胞中受体数量的调节

• 批准号: 7192801
• 负责人: PATRICIA M. HINKLE
• 金额: $ 36.58万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7192801
• 关键词: Acute; Address; Animals; Anterior Pituitary Gland; Antibodies; Arrestin; Arrestins; Binding; Biological Assay; Bolus Infusion; Cell membrane; Cells; Cultured Cells; Depth; Dimerization; Dissociation; Endocytic Vesicle; Endocytosis; Endosomes; Enzyme-Linked Immunosorbent Assay; G-Protein-Coupled Receptors; Goals; Hormone Receptor; Hormones; Hypothalamic structure; Knockout Mice; Label; Learning; Measures; Melanocytic nevus; Mole the mammal; Mutate; Numbers; Organelles; Output; Pathway interactions; Pattern; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiologic pulse; Physiological; Pituitary Gland; Prolactin; Protein Dephosphorylation; Pulse taking; Rate; Rattus; Receptor Activation; Receptor Signaling; Recycling; Role; Signal Transduction; Signal Transduction Pathway; Site; Staining method; Stains; Surface; Techniques; Temperature; Testing; Thyroid Gland; Thyrotropin; Thyrotropin-Releasing Hormone; Thyrotropin-Releasing Hormone Receptors; Tissues; arrestin3; beta-arrestin; desensitization; immunocytochemistry; in vivo; inorganic phosphate; mutant; nutrition; receptor; receptor internalization; receptor recycling; research study; response; stoichiometry; trafficking

DESCRIPTION (provided by applicant): Hypothalamic thyrotropin-releasing hormone (TRH) stimulates thyrotropin and prolactin release by the anterior pituitary gland. The mechanisms of inactivation of the TRH receptor, a Ca2+mobilizing G protein-coupled receptor, are not well understood. Desensitization results from receptor phosphorylation, beta-arrestin binding, and internalization. Resensitization requires receptor dephosphorylation and recycling. There is virtually no information about how the TRH receptor is actually used in vivo. We have recently developed a highly selective phospho-site specific antibody against the phosphorylated (i.e. recently activated) TRH receptor that allows us to address these issues. The phosphorylation sites recognized by the antibody are critical for receptor internalization and desensitization, and the antibody gives strong staining of pituitary tissue from TRH-injected rats. The goals of the proposal are to characterize TRH receptor phosphorylation, dephosphorylation, recycling and resensitization, and to establish how receptor activation occurs in vivo. The first aim uses newly available techniques to determine the stoichiometry of phosphorylation. The kinases involved will be identified, as will the phosphorylation sites on the receptor, which will then be mutated and the effects on signaling, trafficking and desensitization assessed. The next aim will test the hypothesis that dephosphorylation is critical for controlling the intracellular traffic of the internalized receptor. Rates of dephosphorylation of receptors on the plasma membrane and in endocytic vesicles will be measured, and the subcellular sites of dephosphorylation will be determined. The third aim tests the hypothesis that TRH receptor cycling and resensitization are controlled by phosphorylation-dependent beta-arrestin binding. The pathway of TRH receptor recycling will be determined. The role of beta-arrestin in TRH receptor cycling and resensitization will be defined, as will the importance of receptor dimerization. Most experiments in these aims will be done using pituitary GH3 cells. The last aim capitalizes on the ability of the phospho-site specific antibody to identify recently activated TRH receptor. The antibody will be used to follow receptor phosphorylation following a bolus of TRH in rats and in TRH and TRH receptor knockout mice, and to test the hypothesis that hypothalamic TRH drive is responsible for changes in TSH output in vivo in response to changes in thyroid status, nutrition and temperature.

描述（由申请人提供）：下丘脑甲状腺蛋白释放激素（TRH）可刺激垂体前垂体刺激甲状腺激素和催乳素释放。 TRH受体失活的机制，一种Ca2+动员G蛋白偶联受体的机制尚不清楚。脱敏是由受体磷酸化，β-arrestin结合和内在化引起的。复敏需要受体去磷酸化和回收利用。几乎没有关于如何实际在体内使用TRH受体的信息。最近，我们开发了一种针对磷酸化（即最近激活）TRH受体的高度选择性磷酸位点特异性抗体，使我们能够解决这些问题。抗体识别的磷酸化位点对于受体内在化和脱敏至关重要，并且抗体对注入TRH的大鼠的垂体组织染色很强。该提案的目标是表征TRH受体磷酸化，去磷酸化，回收和敏化，并确定体内受体激活的方式。第一个目的使用新近可用的技术来确定磷酸化的化学计量。涉及的激酶将被鉴定出来，受体上的磷酸化位点也将被突变，并评估了对信号，运输和脱敏化的影响。下一个目标将检验以下假设：去磷酸化对于控制内部受体的细胞内流量至关重要。将测量受体在质膜上的受体去磷酸化速率，并将测量脱磷酸化的亚细胞位点。第三个目标检验了TRH受体循环和脱敏化的假设由磷酸化依赖性β-arrestin结合控制。将确定TRH受体回收的途径。 β-arrestin在TRH受体循环和脱敏化中的作用将被定义，受体二聚体的重要性也将定义。这些目标中的大多数实验都将使用垂体GH3细胞进行。最后的目标利用了磷酸位点特异性抗体鉴定最近激活的TRH受体的能力。该抗体将用于跟随大鼠和TRH和TRH受体敲除小鼠的TRH大麻后跟随受体磷酸化，并测试下丘脑TRH驱动器的假设是响应于甲状腺，营养，营养和温度的变化的下丘脑TRH驱动因素负责体内TSH输出的变化。